Mollusk allergy: Not simply cross‐reactivity with crustacean allergens
Authors: Sandip D. Kamath, Tiange Liu, Paul Giacomin, Alex Loukas, Severine Navarro, Andreas L. Lopata
Journal: Allergy (2022)

Mollusk allergy is commonly thought of as clinical cross-reactivity after primary sensitization to shrimps, other crustaceans, or mites.1, 2 Tropomyosin is the major allergen, with primary IgE sensitization in 70% of all shellfish allergies.3 A high frequency of IgE and basophil reactivity to several mollusk allergens is seen in crustacean and mite-sensitized patients.4, 5 It is still unclear, however, whether mollusks are capable of producing primary allergic sensitization, or whether IgE reactivity is based solely on cross-reactive crustacean-specific antibodies. Here, we demonstrate for the first time that mollusk tropomyosin can independently elicit a strong primary T-helper Type 2 (Th2)-mediated IgE response in a IP-sensitized mouse model (BALB/c) of food allergy, which was primarily due to tropomyosin, without any prior sensitization to crustacean allergens. Heated protein extracts were prepared from Jade tiger abalone (hAbal; Haliotis laevigata x Haliotis rubra) and Black tiger shrimp (hBTS; Penaeus monodon). The respective tropomyosins were produced recombinantly (rHal l1 and rPen m1) (Appendix S1). Both proteins share 63% amino acid sequence identity (Figure 1A). Hal l 1 is closely related to oyster and octopus tropomyosin (Figure 1C). In vitro IgE binding was observed to native tropomyosin in hAbal extract (58%) and hBTS extract (75%) as well as for both recombinant tropomyosins, rHal l 1 (62%), and rPen m 1 (75%) as shown by immunoblotting using sera from 12 clinically confirmed shellfish allergic patients (Figure 1B, Tables S1 and S2). Although mono-sensitization to abalone extract or tropomyosin was not observed, subjects 6 and 7 elicited stronger IgE binding to abalone extract and Hal l 1 than to shrimp extracts, implying primary sensitization to abalone. Written informed consent was obtained from all participants, and patient anonymity was preserved. Ethics approval was obtained from the Ethics Committees of James Cook University (H4313), the Alfred Hospital (192/07), and the Monash University (MUHREC CF08/0225). We established two independent mouse models: (i) shrimp allergen model; intraperitoneal-sensitized either with hBTS or rPen m1, and orally challenged with hBTS (Figure 1D); and (ii) abalone allergen model; sensitized either with hAbal extract or rHal l1, and challenged with hAbal. Mouse IgG antibodies produced against abalone tropomyosin sensitization model elicited cross-binding to tropomyosin in the shrimp extract. Likewise, IgE antibodies produced in shrimp-sensitized models showed binding to tropomyosin in the abalone extract (Figure 1E), demonstrating the cross-reactive nature of Hal l 1 and Pen m 1. A strong IgE response to hBTS was elicited (total and shrimp-specific) with a significant release of mast cell protease (mMCP-1) as compared to the naïve group (Figure 2A,B). A similar response was observed with abalone, however, demonstrating relatively stronger IgE and mMCP-1 responses in ELISA. Mice sensitized to Pen m1 or Hal l1 elicited a much stronger IgG1 antibody response than whole extract-sensitized animals, indicating a more efficient Th2 priming potential with purified allergens (Figure 2B). No significant IFN-γ responses confirmed that no Th1 priming occurred (Figure 2C). Antigen-specific in vitro restimulation of splenocytes revealed a robust IL-5 and IL-13 response, but not IFN-γ, indicating a systemic Th2-polarized immune response (Figure 2D). A significant increase in the frequency of blood- and spleen-based IgE+ basophils was observed for abalone- and Hal l1-sensitized mice, similar to shrimp-sensitized mice (Figure 2E). This study demonstrates that abalone tropomyosin is the major sensitizer in the heated extract. Antibodies generated against abalone tropomyosin is capable of binding to shrimp tropomyosin. We recently showed that T-cell cross-reactivity among tropomyosins is not dependent on amino acid sequence similarity, but instead as a function of structural stability.6 This supports our findings that tropomyosins from different invertebrate sources may produce cross-reactive antibodies, even when sharing low amino acid sequence similarity7 Our findings suggest that primary sensitization to tropomyosin from mollusk is possible, and inverse clinical cross-reactivity may occur to crustaceans in mollusk-allergic individuals. SK was supported by a National Health and Medical Research Council early career research fellowship (GNT1124143). SN is supported by a Children's Hospital Foundation grant (RCP10317). ALL was supported by a National Health and Medical Research Council project grant (GNT1086656). The authors have no conflict of interest in relation to this study. The authors thank Prof. Robyn O'Hehir for providing the allergic patient sera. Open access publishing facilitated by James Cook University, as part of the Wiley - James Cook University agreement via the Council of Australian University Librarians. Appendix S1 Table S1 Table S2 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.